Transient receptor potential vanilloid (TRPV) channels are a family of nonselective cation channels that are expressed in skin, neuronal, and non-neuronal cells, and act as polymodal sensors for physicochemical changes in the environment, such as heat, endogenous or exogenous small molecules, and lipids. The thermoTRPV family is composed of four members (TRPV1-TRPV4), each displaying unique functional properties and modalities despite high sequence homology. The distinct features that govern these different modalities have been the subject of intense genetic, biophysical, and physiological studies. TRPV3, which is expressed at high levels in skin keratinocytes, plays vital roles in epidermal homeostasis, hair growth, and skin sensory functions. Precise regulation of this channel's activity is physiologically critical, as gain-of-function mutations (e.g. Gly573Ser) of the trpv3 gene in human and mice lead to itchy dermatitis and hairless phenotypes, while deletion of the gene leads to itch suppression and impaired skin growth and maintenance. TRPV3 inhibitors are currently being developed to relieve pruriceptive pruritus, inflammatory pain, and dermatological disorders, with one candidate in clinical trial. Biophysically, TRPV3 is distinctive amongst thermoTRPV channels in that TRPV3 sensitizes, rather than desensitizes, upon repeated application of stimuli. Despite the recent progress in structural studies of TRP channels, the structure of TRPV3 is unknown, and thus our understanding of the unusual molecular mechanisms of TRPV3 gating has been limited. Importantly, the unique sensitization properties of TRPV3 provide a unique opportunity to observe the open, sensitized channel conformation, which is not feasible using other thermoTRPs. The goal of the proposed research is to describe, at an atomic level, how the human TRPV3 channel responds to chemical stimuli, and how structural rearrangements are coordinated with channel opening. Our preliminary data suggest that conformational rearrangements within the ring of cytoplasmic ankyrin repeat domains (ARDs) are directly linked to TRPV3 channel sensitization and activation. Using a combination of cryo-electron microscopy, mutagenesis, and electrophysiology, we will test this hypothesis in TRPV3 and probe its mechanistic similarity and difference across the thermoTRPV family of ion channels through comparisons with TRPV1 and TRPV2, which desensitize upon repeated stimuli. Successful completion of our aims will provide atomistic descriptions of TRPV3 channel gating, serving as a platform for the development of future drugs targeting human TRPV3. Furthermore, our results will contribute to a general understanding of the gating mechanisms of thermoTRPV channels; importantly explaining how the ARDs mechanistically influence thermoTRPV channel opening, a question that remains a mystery in the field.